Genetic differences exist among individual animals as well as among breeds which can be exploited by breeding techniques to achieve animals with desirable characteristics. For example, Chinese pig breeds are known for reaching puberty at an early age and for their large litter size, while American breeds are known for their greater growth rates and leanness. Often, however, heritability for desired traits is low, and standard breeding methods which select individuals based upon phenotypic variations do not take fully into account genetic variability or complex gene interactions which exist.
There is a continuing need for an approach that deals with selection for disease resistance at the cellular or DNA level. This method will provide the ability to genetically evaluate animals and to enable breeders to more accurately select those animals which not only phenotypically express desirable traits but those which express favorable underlying genetic criteria. This has largely been accomplished to date by marker-assisted selection.
RFLP analysis has been used by several groups to study pig DNA. Jung et al., Theor. Appl. Genet., 77:271-274 (1989), incorporated herein by reference, discloses the use of RFLP techniques to show genetic variability between two pig breeds. Polymorphism was demonstrated for swine leukocyte antigen (SLA) Class I genes in these breeds. Hoganson et al., Abstract for Annual Meeting of Midwestern Section of the American Society of Animal Science, Mar. 26-28, 1990, incorporated herein by reference, reports on the polymorphism of swine major histocompatibility complex (MHC) genes for Chinese pigs, also demonstrated by RFLP analysis. Jung et al. Animal Genetics, 26:79-91 (1989), incorporated herein by reference, reports on RFLP analysis of SLA Class I genes in certain boars. The authors state that the results suggest that there may be an association between swine SLA/MHC Class I genes and production and performance traits. They further state that the use of SLA Class I restriction fragments, as genetic markers, may have potential in the future for improving pig growth performance.
The ability to follow a specific favorable genetic allele involves a novel and lengthy process of the identification of a DNA molecular marker for a major effect gene. The marker may be linked to a single gene with a major effect or linked to a number of genes with additive effects. DNA markers have several advantages; segregation is easy to measure and is unambiguous, and DNA markers are co-dominant, i.e., heterozygous and homozygous animals can be distinctively identified. Once a marker system is established, selection decisions could be made very easily, since DNA markers can be assayed any time after a tissue or blood sample can be collected from the individual infant animal, or even an embryo.
The use of genetic differences in receptor genes has become a valuable marker system for selection. For example, U.S. Pat. Nos. 5,550,024 and 5,374,526, issued to Rothschild et al., disclose a polymorphism in the pig estrogen receptor gene which is associated with larger litter size, the disclosure of which is incorporated herein by reference. U.S. Pat. No. 5,935,784 discloses polymorphic markers in the pig prolactin receptor gene which are associated with larger litter size and overall reproductive efficiency, the disclosure of which is incorporated herein by reference.
NRAMP1 Gene
The NRAMP1 gene was isolated from a murine Bcg candidate gene and designated “natural resistance-associated macrophage protein” gene by Vidal and coworkers (Vidal, et al., Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell 73, 469-485 (1993)). Bcg was found during genetic studies of mice to mediate antimicrobial activity of macrophages against intracellular parasites early during infection. The isolated NRAMP1 gene apparently encodes an integral membrane protein that has structural features similar to prokaryotic and eukaryotic ion transporters. More recent studies using knockout mice (Vidal, et al., J. Exp. Med. 182:655-666 (1995)) indicated the NRAMP1 is the Bcg/Lsh/Ity gene (3 genes capable of controlling resistance and susceptibility to Mycobacterium bovis (BCG), Leishmania donovani and Salmonella typhimurium, respectively, known genetically to be a single gene expressed at the macrophage level, Blackwell, J. M. The macrophage resistance gene Lsh/Ity/Bcg. Res. Immunol. 140: 767 (1989)). It has been suggested that the murine NRAMP1 protein might function in phagolysosomal membranes as a concentrator of nitric oxide, mediating cytocidal activity against the ingested parasites of infected macrophage (Vidal, et al. 1993; Malo, et al. Genetic control of host resistance to infection. TIG 10, 365-371 (1994); Cellier, et al. Human natural resistance-associated macrophage protein: cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression. J. Exp. Med. 180, 1741-1752 (1994); Malo, et al Haplotype mapping and sequence analysis of the mouse NRAMP1 gene predicts susceptibility to infection with intracellular parasites. Genomics 23, 51-61 (1994)). It has recently been indicated that the mammalian NRAMP protein family (at least NRAMP 2) functions as broad specificity divalent cation transporters (Gunshin, et al. Nature 388: 482, 1997; Fleming, et al. Nature Genet. 16: 383, 1997). A cDNA for NRAMP1 was isolated from a pre B-cell cDNA library and sequenced. The amino acid sequence for the protein product was deduced from the nucleotide sequence and predicts a 53 kDa protein. On the basis of the deduced amino acid sequence, Vidal et al. (1993) proposed as a function of the NRAMP1 protein the transport of nitrate across the membrane of the intracellular vacuole of the macrophage containing the microorganisms. In the acid environment of this vacuole, the nitrate could be converted via nitrite to toxic nitric oxide thereby enhancing killing of the microorganisms. The NRAMP1 protein has been localized to the phagolysosomal membrane, and with the data on NRAMP2 function, an alternative function has recently been proposed. It is known that bacteria use superoxide dismutase (SOD) to detoxify the phagolysosome. SOD requires divalent cations, notably Mn++. It is proposed that NRAMP1 may pump metal ions (such as Fe++ or Mn++) out of the phagolysosome compartment, thus depriving bacteria of this defense mechanism (G. Govoni and P. Gros (1998) Inflamm. Res. 47:277-284).
Nucleotide sequence analyses of murine NRAMP1 cDNA showed that the susceptible phenotype was associated with a nonconservative glycine-to-aspartic acid amino acid substitution within the second trans membrane domain of the protein (Vidal, et al. 1993).
Whatever the mechanism of NRAMP1, it has been recognized as important for its role in infection resistance in several other species, in addition to mice. Its homologues, variants and polymorphisms have been investigated in humans and agriculturally important animals, with unpredictable results for use of these variants as markers of any phenotype. For example, see, international application WO 95/13371 to Gros et al.
Gros et al. discloses mouse and human cDNAs from NRAMP1 genes. The nucleotide sequences of mouse and human cDNAs are disclosed, as are the amino acid sequences. Throughout the document a potential use for the nucleotide sequences is disclosed as assaying for different mutations or variability which is associated with susceptibility or resistance to infectious diseases including tuberculosis, leprosy, salmonellosis, and leshmaniasis.
The application discloses isolation of the mouse gene from a library and the identification of two alternate forms Bcgr and Bcgs, which had a single nucleotide difference, namely, a guanine at nucleotide 783, which was present in resistant strains. Importantly, however, there were two additional silent mutations detected in the 5′ portion of the transcript from a resistant mice strain, one at nucleotide position 563 and another at nucleotide position 1169 which did not have any different phenotypic effect. Out of all polymorphisms detected in the tested strains, four out of five polymorphisms identified were silent mutations and were not shown to be associated with any difference in susceptibility to infection.
Application number WO 98/12353 to Templeton et al. discloses identification and sequencing of homologues of murine NRAMP1 from bovine, bison and other artiodactyla. The invention discloses particular sequences of NRAMP1 which correlate with resistance or susceptibility to brucellosis, tuberculosis, paratuberculosis and salmonellosis in cattle. The sequence associated with resistance/susceptibility is a transversion at position 1782 of the NRAMP1 cDNA and a polymorphic DNA microsatellite sequence difference.
The examples section discloses cloning of bovine NRAMP1 and isolation of bovine NRAMP1 cDNA, analysis of predicted bovine NRAMP1 structure, genetic mapping of bovine NRAMP1, single-stranded conformational analysis disclosing the microsatellite length polymorphism, cell specific expression of bovine NRAMP1 mRNA, SSCA disclosing a 3′ untranslated region polymorphism, and association of resistances or susceptibility to ruminant brucellosis, tuberculosis, paratuberculosis and salmonellosis, with the alternative gene forms.
The Barton et al. application, WO 95/20044, discloses the sequence of NRAMP1 gene isolated from mice. Function and uses of the gene include diagnosing the susceptibility or resistance to microorganisms including Salmonella typhimurium, Leishmania donovani and Mycobacterium bovis. A resistant allele of murine NRAMP1 was found with two silent mutations, one at 359 base pairs and one at 965 base pairs. In humans a polymorphic repeat in the 5′ promoter region was identified, with no association with a useful trait disclosed.
The Rotter et al. reference (WO 99/23255) relates to a method of identifying novel alleles or allelic combinations in the human NRAMP1 locus which evidence statistically significant correlation with one or more biological responses (humans with diseases), such as Crohn's Disease, Ulcerative Colitis or their subtypes. Seven alleles of the NRAMP1 satellite marker were studied. The D2S434 allele is characterized by seven different fragment sizes from 262 base pairs to 286 base pairs characterized by repeats of GATA(N). The 2DS1323 NRAMP1 allele was identified in two forms of GATA(N) repeats, one of 324 base pairs and one of 328 base pairs. The D2S1323 polymorphism and D2S434 polymorphism were analyzed for linkage to various diseases. Three mutated and wild type NRAMP1 DNA sequences were also obtained from patients, and associations for some of the alleles were found with inflammatory bowel disease and ulcerative colitis.
As can be seen, the NRAMP1 gene is known to be variable and it is highly unpredictable as to which if any variations are associated with useful traits to be of value as a genetic marker.
The present invention provides a genetic markers, based upon the discovery of a polymorphisms in the porcine NRAMP1 gene, which correlate with resistance or susceptibility to pathogenic infection in pigs. This will permit genetic typing of pigs for their NRAMP1 allele and for determination of the relationship of specific RFLPs to resistance to infection. It will also permit the identification of individual males and females that carry the gene for improved resistance. Thus, the markers may be selection tools in breeding programs to develop lines and breeds that produce litters containing more resistant offspring. Also disclosed are novel porcine NRAMP1 genomic sequences, as well as primers for assays to identify the presence or absence of marker alleles.
According to the invention a polymorphism was identified in the NRAMP1 gene which is associated with the improved resistance to pathogenic infection.
It is an object of the invention to provide a method of screening pigs to determine those more likely to produce offspring with improved pathogenic resistance, in the NRAMP1 gene.
Another object of the invention is to provide a method for identifying genetic markers for improved disease resistance.
A further object of the invention is to provide genetic markers for selection and breeding to obtain pigs that will be expected to have a lower susceptibility to infection than those without the favorable allele.
Yet another object of the invention is to provide a kit for evaluating a sample of pig DNA for specific genetic markers of disease resistance.
Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objects and advantages of the invention will be attained by means of the instrumentality's and combinations particularly pointed out in the appended claims.